Method for the preparation of chromanone 7

ABSTRACT

A method of preparing (±)-calanolide A, 1, a potent HIV reverse transcriptase inhibitor, from chromene 4 is provided. Useful intermediates for preparing (±)-calanolide A and its derivatives are also provided. According to the disclosed method, chromene 4 intermediate was reacted with acetaldehyde diethyl acetal or paraldehyde in the presence of an acid catalyst with heating, or a two-step reaction including an aldol reaction with acetaldehyde and cyclization either under acidic conditions or neutral Mitsunobu conditions, to produce chromanone 7. Reduction of chromanone 7 with sodium borohydride, in the presence of cerium trichloride, produced (±)-calanolide A. A method for resolving (±)-calanolide A into its optically active forms by a chiral HPLC system or by enzymatic acylation and hydrolysis is also disclosed. Finally, a method for treating or preventing viral infections using (±)-calanolide A or (-)-calanolide A is provided.

CROSS-REFERENCE

This is a divisional of application Ser. No. 08/510,213, filed Aug. 2,1995, which in turn is a continuation-in-part of U.S. patent applicationSer. No. 08/285,655, filed Aug. 3, 1994, now U.S. Pat. No. 5,489,697,issued Feb. 6, 1996.

FIELD OF THE INVENTION

This invention relates to a method for the preparation of (±)-calanolideA, a potent inhibitor of HIV reverse transcriptase, and intermediatesthereof. In particular, this invention relates to a method for largescale production of (±)-calanolide A, chiral resolution of(±)-calanolide A into its optically active forms, and use of(±)-calanolide A and (-)-calanolide A for treating viral infections.

BACKGROUND OF THE INVENTION

Human immunodeficiency virus (HIV), which is also called humanT-lymphotropic virus type III (HTLV-III), lymphadenopathy-associatedvirus (LAV) or AIDS-associated retrovirus (ARV), was first isolated in1982 and has been identified as the etiologic agent of the acquiredimmunodeficiency syndrome (AIDS) and related diseases. Since then,chemotherapy of AIDS has been one of the most challenging scientificendeavors. So far, AZT, ddC, ddI, and D4T have been approved by FDA andare being clinically used as drugs for the treatment of AIDS andAIDS-related complex. Although these FDA-approved drugs can extend thelife of AIDS patients and improve their quality of life, none of thesedrugs are capable of curing the disease. Bone-marrow toxicity and otherside effects as well as the emergence of drug-resistant viral strainslimit the long-term use of these agents.¹ On the other hand, the numberof AIDS patients worldwide has increased dramatically within the pastdecade and estimates of the reported cases in the very near future alsocontinue to rise dramatically. It is therefore apparent that there is agreat need for other promising drugs having improved selectivity andactivity to combat AIDS.¹ Several approaches including chemicalsynthesis, natural products screening, and biotechnology have beenutilized to identify compounds targeting different stages of HIVreplication for therapeutic intervention.²

Very recently, the screening program at the National Cancer Institutehas discovered a class of remarkably effective anti-HIV naturalproducts, named calanolides, from the rain forest tree Calophyllumlanigerum, with calanolide A, 1, being the most potent compound in thereported series.³ For example, calanolide A demonstrated 100% protectionagainst the cytopathic effects of HIV-1, one of two distinct types ofHIV, down to a concentration of 0.1 μM. This agent also halted HIV-1replication in human T-lymphoblastic cells (CEM-SS)(EC₅₀ =0.1 μM/IC₅₀=20 μM).³ More interestingly and importantly, calanolide A was found tobe active against both the AZT-resistant G-9106 strain of HIV as well asthe pyridinone-resistant A17 virus.³ Thus, the calanolides, known asHIV-1 specific reverse transcriptase inhibitors, represent novelanti-HIV chemotherapeutic agents for drug development.

A source of calanolide A is limited. Consequently, a practical synthesisof the natural product must be developed for further study anddevelopment to be carried out on this active and promising series ofcompounds. Herein, we describe a method for the synthesis and resolutionof (±)-calanolide A and some related compounds. ##STR1##

OBJECTS OF THE INVENTION

Accordingly, one object of the present invention is to provide a simpleand practical method for preparing (±)-calanolide A, 1, from readilyavailable starting materials and resolving the same into its opticallyactive forms via a chiral HPLC system or enzymatic acylation andhydrolysis.

Another object of the invention is to provide useful intermediates forpreparing derivatives of (±)-calanolide A.

A further object of the invention is to provide a simple and practicalmethod for large scale preparation of (±)-calanolide A in high yieldsfrom key intermediate chromene 4.

An additional object of the invention is to provide a method fortreating or preventing viral infections using (±)-calanolide A and(-)-calanolide A.

These and other objects of the invention will become apparent in view ofthe detailed description below.

SUMMARY OF THE INVENTION

The present invention relates to the syntheses of (±)-calanolide A andintermediates thereof, chiral resolution of (±)-calanolide and method oftreating or preventing viral infections using (±)- and (-)-calanolide A.

The method of the present invention for preparing (±)-calanolide A, 1,employs chromene 4 as the key intermediate. Chromene 4 is synthesized bythe sequence depicted in Scheme I. Thus, 5,7-dihydroxy-4-propylcoumarin,2,5 was prepared quantitatively from ethyl butyrylacetate andphloroglucinol under Pechmann conditions.⁶ Product yield and purity weredependent on the amount of sulfuric acid used. The 8-position of5,7-dihydroxy-4-propylcoumarin, 2, was then selectively acylated at8°-10° C. by propionyl chloride and AlCl₃ in a mixture of carbondisulfide and nitrobenzene to afford5,7-dihydroxy-8-propionyl-4-propylcoumarin, 3.

In an alternative and preferred reaction, coumarin intermediate 3 can beproduced in large scale quantities and with minimal formation ofundesirable 6-position acylated product and 6,8-bis-acylated product byselective acylation of 5,7-dihydroxy-4-propylcoumarin 2 with a mixtureof propionic anhydride and AlCl₃ at about 70°-75° C.

The chromene ring was introduced upon treatment of compound 3 with4,4-dimethoxy-2-methylbutan-2-ol,⁸ providing 4 in 78% yield Scheme I.

As presented in Scheme II, Robinson-Kostanecki reaction⁹ on 4 by usingsodium acetate in refluxing acetic anhydride produced enone 5 in a 65%yield. This intermediate failed to afford calanolide A upon reductionwith borohydride reagents such as NaBH₄ /CeCl₃, NaBH₄ /CuCl₂,L-selectride, 9-BBN, and DIP-chloride, and some transition metalreducing agents such as SmI₂ and (Ph₃ P)CuH!₆, presumably because attackat the pyrone and ring opening occurred preferentially. Treatment of 5with Baker's yeast also resulted in coumarin ring cleavage whiletri-n-butyltin hydride¹⁰ led to reduction of 5 to enol 6 in modestyield. However, treatment of 4 with acetaldehyde diethyl acetal in thepresence of trifluoroacetic acid and pyridine with heating at 160° C.produced chromanone 7 which can then be reduced into the final product.##STR2##

Large scale production of chromanone 7 from chromene 4 can be effectedunder two different reactions conditions. In a one-step reaction,chromene 4 is treated with paraldehyde, instead of acetaldehydediethylacetal, and cyclized in the presence of an acid catalyst toafford chromanone 7 in 27% yield along with 8% of the corresponding10,11-cis-dimethyl derivative 7a. In a two-step reaction under aldolcondensation conditions, chromene 4 was reacted with acetaldehyde toform an open-chain aldol product 7b. Aldol product 7b was then cyclizedunder acidic conditions such as 50% H₂ SO₄ and TsOH to form bothchromanone 7 and 10,11-cis-dimethyl derivative 7a in a 1:1 ratio withthe former leading to 16% purified yield. However, under neutralMitsunobu¹¹ conditions, 7b was reproducibly cyclized to give chromanone7 as the predominant product and in 48% yield. ##STR3##

Finally, (±)-calanolide A was successfully formed with the desiredstereochemical arrangement by subjecting chromanone 7 to Luche reduction¹² conditions (see Scheme II). (±)-Calanolide A was then resolved intooptically active forms using a preparative HPLC chiral separatingsystem¹³.

DESCRIPTION OF THE DRAWINGS

FIGS. 1(a) to 1(e) illustrate in vitro MTT assay results, as describedin Example 15, using G9106 HIV viral strain which is AZT-resistant.

FIGS. 2(a) to 2(e) illustrate in vitro MTT assay results, as describedin Example 15, using H112-2 HIV viral strain which was not pre-treatedwith AZT.

FIGS. 3(a) to 3(e) illustrate in vitro MTT assay results, as describedin Example 15, using A-17 HIV viral strain which is resistant tonon-nucleoside inhibitors such as TIBO and pyridinone but is sensitiveto AZT.

FIGS. 4(a) to 4(d) illustrate in vitro MTT assay results, as describedin Example 15, using IIIB cultivated HIV viral strain.

FIGS. 5(a) to 5(d) illustrate in vitro MTT assay results, as describedin Example 15, using RF cultivated HIV viral strain.

FIG. 6 is an HPLC chromatogram of (a) (±) calanolide A on normal phasecolumn; (b) (±)-calanolide A on a chiral HPLC column; (c) (+)-calanolideA on a chiral HPLC column and (d) (-)-calanolide A on on a chiral HPLCcolumn. The HPLC conditions are described in Example 13.

DETAILED DESCRIPTION OP THE INVENTION

All patents, patent applications, and literature references cited hereinare incorporated by reference in their entirety.

According to the method of the present invention, chromene 4 is a keyintermediate in the preparation of (±)-calanolide A, 1. A preferredmethod for synthesizing chromene 4 from 5,7-dihydroxy-4-propylcoumarin,2, is shown in Scheme I. According to this synthetic scheme,5,7-dihydroxy-4-propylcoumarin, 2,⁵ was prepared quantitatively fromethyl butyrylacetate and phloroglucinol under Pechmann conditions.⁶

In conducting this reaction, a volume of a concentrated acid is added ina dropwise manner to a stirring mixture of ethyl butyrylacetate andphloroglucinol with a mole ratio ranging between about 3:1 and about1:3, with a preferable range being about 0.9:1.0. The dropwise additionof an acid was conducted at a rate such that the temperature of thereaction mixture is maintained at a temperature ranging between about 0°C. and about 120° C., preferably about 90° C.

Suitable, but not limiting, examples of concentrated acid includesulfuric acid, trifluoroacetic acid, and methanesulfonic acid. Inpracticing this invention, concentrated sulfuric acid is particularlypreferred. As the product yield and purity appear to be dependent on theamount of concentrated sulfuric acid used, it is preferred that theamount of concentrated sulfuric acid range between about 0.5 and 10moles, most preferably ranging between about 2 and about 3.5 moles, permole of ethyl butyrylacetate.

The reaction mixture is then heated to a temperature ranging betweenabout 40° C. and about 150° C., preferably about 90° C., until thereaction reaches completion as determined by TLC analysis. The reactionmixture is then poured onto ice and the precipitated product iscollected by filtration and dissolved in an organic solvent. Suitable,but non-limiting, examples of organic solvents include ethyl acetate,chloroform, and tetrahydrofuran. A preferred solvent is ethyl acetate.The resulting solution is then washed with brine and dried over asuitable drying agent, e.g., sodium sulfate. The yields of this reactionare generally quantitative.

Thereafter, 5,7-dihydroxy-8-propionyl-4-propylcoumarin, 3, was preparedby selectively acylating the 8-position of5,7-diydorxy-4-propylcoumarin, 2, with propionyl chloride in thepresence of a Lewis acid catalyst. In conducting this reaction, asolution of propionyl chloride in a suitable solvent, e.g., carbondisulfide, was added in a dropwise manner to a vigorously stirredsolution of 5,7-dihydroxy-4-propylcoumarin, 2, a Lewis acid and anorganic solvent cooled in an ice bath. Dropwise addition of propionylchloride is conducted such that the temperature of the reaction mixtureis maintained at a temperature ranging between 0° C. and about 30° C.,preferably between about 8° C. and 10° C.

In practicing the invention, the amount of propionyl chloride usedgenerally ranges between about 0.5 moles and about 6 moles, preferablyranging between about 1 mole and about 2 moles, per mole of5,7-dihydroxy-4-propylcoumarin, 2.

Non-limiting examples of Lewis acid catalysts useful in the acylationreaction include AlCl₃, BF₃, SnCl₄, ZnCl₂, POCl₃ and TiCl₄. A preferredLewis acid catalyst is AlCl₃. The amount of Lewis acid catalyst relativeto 5,7-dihydroxy-4-propylcoumarin, 2, ranges between about 0.5 and about12 moles, preferably ranging between about 2 and about 5 moles, per moleof 5,7-dihydroxy-4-propylcoumarin, 2.

Non-limiting examples of organic solvent for use in preparing the5,7-dihydroxy-4-propylcoumarin, 2, solution include nitrobenzene,nitromethane, chlorobenzene, or toluene and mixtures thereof. Apreferred organic solvent for use in this invention is nitrobenzene.

Upon completion of the addition of propionyl chloride, the vigorouslystirred reaction mixture is maintained at a temperature ranging betweenabout 0° C. and about 120° C., preferably ranging between about 25° C.and 80° C., until the reaction reaches completion as monitored byconventional means such as TLC analysis. The reaction mixture is thenpoured onto ice and extracted several times with a suitable solvent suchas ethyl acetate, chloroform, methylene chloride, tetrahydrofuran, or amixture of chloroform/methanol. A preferred solvent for this extractionis ethyl acetate. The extracts are then dried over a suitable dryingagent, e.g., sodium sulfate, and the product may be purified byconventional means such as silica gel column chromatography.

On a small scale, the yield of5,7-dihydroxy-8-propionyl-4-propylcoumarin 3, produced by the abovedescribed reaction is generally quantitative. However, on a largerscale, the reaction was very difficult to control and did notexclusively afford the desired product. A route developed for thesynthesis of Mammea coumarin was initially attempted for the preparationof compound 3, but it proved too awkward and low-yielding.⁷

Since the desired 8-position acylated product 3 was always accompaniedby the formation of undesired 6-position acylated product and6,8-bis-acylated product, it was necessary to optimize the reactionconditions to minimize the formation of the undesired products anddevelop a more effective purification process to increase the purity andscale-up the quantities of the desired5,7-dihydroxy-8-propionyl-4-propylcoumarin 3. An alternative andpreferred route for preparing 5,7-dihydroxy-8-propionyl-4-propylcoumarin3 in large scale quantities is then developed.

Preparation of 8-acylated coumarin 3 on a 5 gram scale as a singleproduct (45% yield) has been achieved by adding a mixture of propionicanhydride, a Lewis acid, e.g., AlCl₃, and suitable solvent, e.g.,1,2-dichloroethane, into a vigorously stirring pre-heated mixture ofcoumarin, a Lewis acid, e.g., AlCl₃, and suitable solvent, e.g.,1,2-dichloroethane, at a temperature ranging between about 40° and about160° C., preferably ranging between about 70° and about 75° C. Dropwiseaddition of the propionic anhydride solution is conducted at a rate suchthat the temperature of the reaction mixture is maintained within thedesired temperature range.

The amount of propionic anhydride used in the reaction generally rangesbetween about 0.5 and about 10 moles, preferably ranging between about 1and about 2 moles, per mole of 5,7-dihydroxy-4-propylcoumarin 2.

Non-limiting examples of Lewis acid catalysts useful in the acylationreaction include AlCl₃, BF₃, POCl₃, SnCl₄, ZnCl₂ and TiCl₄. A preferredLewis acid catalyst is AlCl₃. The amount of Lewis acid catalyst relativeto 5,7-dihydroxy-4-propylcoumarin, 2, ranges between about 0.5 and about12 moles, preferably ranging between about 2 and about 4 moles, per moleof 5,7-dihydroxy-4-propylcoumarin, 2.

Suitable but nonlimiting examples of solvents for use in the inventioninclude diglyme, nitromethane, 1,1,2, 2-tetrachloroethane, and1,2-dichloroethane (preferred).

Upon completion of the addition of propionyl anhydride, the vigorouslystirred reaction mixture is maintained at a temperature ranging betweenabout 40° C. and about 160° C., preferably ranging between about 70° C.and 75° C., until the reaction reaches completion as monitored byconventional means such as TLC analysis. The workup procedure is thesame as described above.

The product was purified without the use of column chromatography toafford the desired product 3. This procedure has been scaled-up to 1.7kg of coumarin (for details see experimental section) and the yield for8-acylated coumarin 3 was 29% after recrystallization. The yield for8-acylated coumarin 3 may be further improved by changing thepurification processing. For example, the crude product may berecrystallized from solvent(s) other than dioxane, or a simple washingwith an appropriate solvent may lead to product pure enough for the nextreaction step.

Thereafter, chromene 4 was prepared by introducing the chromene ringinto 5,7-dihydroxy-8-propionyl-4-propylcoumarin, 3, using4,4-dimethoxy-2-methylbutan-2-ol. According to the method of the presentinvention, a solution of 5,7-dihydroxy-8-propionyl-4-propylcoumarin, 3,and 4,4-dimethoxy-2-methylbutan-2-ol in a suitable organic solvent inthe presence of a base was reacted at a temperature ranging betweenabout 40° C. and about 180° C., preferably ranging between about 100° C.and about 120° C., until the reaction reached completion as determinedby conventional means such as TLC analysis. Water and methanol formedduring the reaction were removed azeotropically via a Dean-Stark trap.

In practicing this invention, the amount of4,4-dimethoxy-2-methylbutan-2-ol employed in the reaction generallyranges between about 0.5 and about 8 moles, preferably ranging betweenabout 2 and about 4 moles, per mole of5,7-dihydroxy-8-propionyl-4-propylcoumarin 3.

Suitable, but not limiting examples of organic solvents includepyridine, triethylamine, N,N-dimethylformamide (DMF), toluene,tetrahydrofuran (THF) or 1,2-dichloroethane. Suitable, but non-limitingexamples of the bases include pyridine, 4-dimethylaminopyridine,triethylamine, N,N-diethylaniline, 1,5-diazabicyclo- 4,3,0!-non-5-ene(DBN), 1,8-diazabicyclo- 5,4,0!undec-7-ene (DBU), sodium carbonate andsodium bicarbonate. Pyridine was used as both base and solvent in thisinvention on a small scale; for scale-up, however, pyridine was used asa base and toluene was used as a solvent.

Upon completion of the reaction, the solvent is removed under reducedpressure and the reaction products is dissolved in a suitable solvent,e.g., ethyl acetate. The solution is then washed sequentially with waterand brine and dried over a suitable drying agent, e.g., sodium sulfate.Thereafter, the crude chromene 4 product can be purified by conventionalmeans such as silica gel column chromatography using 25% ethylacetate/hexane as the elution solvent. The yields of chromene 4generally fall with the range of about 60% and about 85%, usuallyresulting in about 78% yield.

Thereafter, chromanone 7 may be produced by reacting a solution ofchromene 4, acetaldehyde diethylacetal, and an acid catalyst in organicsolvent at a temperature ranging between about 60° C. and about 140° C.,preferably about 140° C., until the reaction is completed.

The amount of acetaldehyde diethylacetal used in the reaction generallyranges between about 0.5 and about 20 moles, preferably ranging betweenabout 3 and about 5 moles, per mole of chromene 4.

Suitable, but non-limiting, examples of acid catalysts includetrifluoroacetic acid, methanesulfonic acid, trifluoromethanesulfonicacid, p-tosylic acid, acetic acid, hydrofluoric acid and theirpyridinium salts and mixtures thereof. A preferred acid catalyst for theuse in this invention is trifluoroacetic acid. The amount of acidcatalyst used generally ranges between about 2 and about 25 moles,preferably ranging between about 17 and about 22 moles, per mole ofchromene 4.

Two alternative routes for preparing chromanone 7 from chromene 4 inlarge scale quantities were developed and which involve either aone-step reaction process (paraldehyde one-step reaction) or a two-stepreaction processes (LDA/sulfuric acid process or LDA/Mitsunobu process)

(a) Paraldehyde one-step reaction

Instead of acetaldehyde diethylacetal, paraldehyde was used as theacetaldehyde equivalent. In the presence of one or more acid catalystssuch as CF₃ SO₃ H, CF₃ CO₂ H, and pyridinium p-toluenesulfonate (PPTS),chromene 4 was reacted with paraldehyde at elevated temperature in asuitable solvent to afford chromanone 7 as the major product and thecorresponding 10,11-cis-dimethyl derivative 7a as a minor product.

According to this reaction, paraldehyde was added to a stirring solutionof chromene 4 and an acid catalyst, e.g., PPTS, at room temperature in asuitable solvent. The resulting mixture was heated at the temperatureranging between about 40° and about 140° C., preferably ranging betweenabout 60° and about 100° C., for a period of time ranging between about5 and about 36 hours, preferably about 20 hours. Thereafter, CF₃ CO₂ H,an additional equivalent of PPTS and paraldehyde was added and theresulting mixture was maintained at a temperature ranging between about40° and about 140° C., preferably ranging between about 60° and about100° C. overnight or until the reaction reached completion as determinedby convention means, e.g., TLC.

The amount of paraldehyde employed per mole of chromene 4 generallyranges between about 1 and about 40 moles, preferably ranging betweenabout 20 and about 30 moles.

Non-limiting acid catalysts include trifluoromethane sulfonic acid,trifluoroacetic acid, methanesulfonic acid, p-tosylic acid and theirpyridinium salts. In practicing this invention, pyridiniump-toluenesulfonate (PPTS) is the preferred acid catalyst. The amount ofacid catalyst used in the reaction ranges between about 0.5 and about 10moles, preferably between about 1 and about 2 moles.

Representative solvents for use in the reaction include toluene, diglymeand 1,2-dichloroethane. In practicing the invention, 1,2-dichloroethaneis the preferred solvent.

Upon completion of the reaction, the reaction was neutralized withsaturated bicarbonate solution and extracted with a suitable solvent,e.g., ethyl acetate. The crude product was then purified as describedabove. The yields of chromanone 7 from this reaction generally rangebetween about 20 and about 60%, usually about 40%.

(b) LDA/sulfuric acid two-step reaction

Under aldol condensation conditions, chromene 4 was reacted withacetaldehyde to form an open-chain aldol product 7b. According to thepresent invention, a solution of LDA in THF was added dropwise to asolution of chromene 4 in THF at a temperature ranging between about-78° C. and about 0° C., preferably about -30° C. and about -78° C. Theamount of LDA added per mole of chromene 4 ranged between about 1 andabout 4 moles , preferably ranging between about 2 and about 3 per moleof chromene 4. Dropwise addition LDA is conducted such that the reactiontemperature is maintained within the desired range.

Acetaldehyde was then added dropwise to the reaction mixture in amountsranging between about 1 and about 12 moles, preferably ranging betweenabout 4 and about 6 moles per mole of chromene 4. Dropwise addition ofacetaldehyde is conducted such that the reaction temperature ismaintained within the aforementioned range. The reaction was monitoredby conventional means, e.g., TLC analysis, until it reached completion.

One skilled in the art will appreciate that the aldol reaction ofchromene 4 with acetaldehyde to form 7b can be carried out underconditions which employs bases other than LDA. For example, metalhydroxides such as NaOH, KOH and Ca(OH)₂, metal alkoxides such as MeONa,EtONa and t-BuOK, and amines such as pyrrolidine, piperidine,diisopropylethylamine, 1,5-diazabicyclo 4,3,0!non-5-ene (DBN),1,8-diazabicyclo 5,4,0!undec-7-ene (DBU), LDA, NaNH₂ and LiHMDS as wellas hydrides such as NaH and KH can all be employed for the aldolreactions.¹⁵ Also, aldol reactions can be mediated by metal complexes ofAl, B, Mg, Sn, Ti, Zn and Zr compounds such as TiCl₄, (i-PrO)₃ TiCl,(i-PrO)₄ Ti, PhBCl₂, (n-Bu)₂ BCl, BF₃, (n-Bu)₃ SnCl, SnCl₄, ZnCl₂,MgBr₂, Et₂ AlCl with or without chiral auxiliaries such as1,1'-binaphthol, norephedrinesulfonate, camphanediol, diacetone glucoseand dialkyl tartrate.¹⁶⁻¹⁸

Thereafter, the reaction mixture was quenched at -30° C. to -10° C. withsaturated aqueous ammonium chloride solution and extracted with asuitable solvent, e.g., ethyl acetate. The pooled extracts were washedwith brine and dried over a suitable drying agent, e.g., sodium sulfate.The yields of aldol product 7b generally range between about 40% andabout 80%, usually about 70%.

It should be noted that there are two asymmetric centers in 7b.Therefore, 7b is racemic mixture of two sets of enantiomers (fouroptically active forms) which may be resolved by conventional resolutionmethods such as chromatography or fractional crystallization of suitablediastereoisomeric derivatives such as salts or esters with opticallyactive acids (e.g., camphor-10-sulfonic acid, camphoric acid,methoxyacetic acid, or dibenzoyltartaric acid) or enzymaticallycatalyzed acylation or hydrolysis of the racemic esters. Also, chiraltransition metal-mediated aldol reaction¹⁷,18 of chromene 4 withacetaldehyde may directly produce optically active one of theenantiomers of 7b. The resultant or synthetic enantiomer may then betransformed to enantioselective synthesis of (+)-calanolide A and itscongeners.

The crude aldol product 7b was then cyclized under acidic conditions toform a mixture of both chromanone 7 and 10,11-cis-dimethyl derivative 7ain a 1:1 ratio. Suitable, but non-limiting, acids include one or moreacids such as sulfuric acid, hydrochloric acid (aqueous or anhydrous),trifluoroacetic acid, methanesulfonic acid, trifluoromethanesulfonicacid, p-tosylic acid, acetic acid or their mixture thereof. A preferredacid for use in the reaction is a 1:1 v/v mixture of acetic acid and 50%H₂ SO₄.

The reaction mixture was cooled, ice water was added and the resultingmixture extracted with a suitable solvent, e.g., ethyl acetate. Thepooled organic layers were washed with water, saturated bicarbonatesolution and brine. The crude product was concentrated in vacuo andpurified by conventional means, e.g., silica gel column using a 2:3(v/v) ethyl acetate/hexane solvent mixture. The yields of chromanone 7from this reaction generally range between about 10% and about 40%,usually about 20% based on chromene 4.

One skilled in the are will also appreciate that 10,11-cis-chromanone 7acan be treated with with a base under thermodynamic (equilibrium)conditions so as to afford the corresponding trans-chromanone 7. Thesuitable, but non-limiting, bases include metal hydroxides such as NaOH,KOH and Ca(OH)₂, metal alkoxides such as MeONa, EtONa and t-BuOK, aminessuch as triethylamine, diisopropylethylamine, pyridine,4-dimethylaminopyridine, N,N-diethylaniline, pyrrolidine, piperidine,1,5-diazabicyclo 4,3,0!non-5-ene (DBN), 1,8-diazabicyclo-5,4,0!undec-7-ene (DBU), LDA and LiHMDS as well as metal hydrides suchas NaH and KH.

(c) LDA/Mitsunobu two-step reaction

In a preferred reaction, aldol product 7b may be converted to chromanone7 as the predominant product under neutral Mitsunobu reactionconditions. In this reaction, diethyl azodicarboxylate (DEAD) was addeddropwise to a solution containing crude aldol product 7b andtriphenylphosphine at a temperature ranging between about -10° C. andabout 40° C., preferably about ambient temperature. The amount of DEADused in the reaction generally ranges between about 1 and about 10 molespreferably about 1 and about 4 moles, per mole of aldol 7b. The amountof triphenylphosphine used in the reaction generally ranged betweenabout 1 and about 10 moles, preferably ranging between about 1 and about4 moles, per mole of aldol 7b.

Instead of DEAD, other reagents reported in the literature can beemployed such as diisopropyl azodicarboxylate (DIAD), dibutylazodicarboxylate (DBAD), dipiperidinoazodicarboxamide, bis(N⁴-methylpiperazin-1-yl)azodicarboxamide, dimorpholinoazodicarboxamide,N,N,N',N'-tetramethylazodicarboxamide (TMAD)⁹. Also, in addition totriphenylphosphine, tri-n-butylphosphine,¹⁹ triethylphosphine,trimethylphosphine and tris(dimethylamino)-phosphine have been used.

Thereafter, the reaction was quenched with saturated ammonium chlorideupon completion and extracted with a suitable solvent, e.g., ethylacetate. The pooled organic layers were washed with brine, concentratedin vacuo and the crude chromanone 7 was purified by conventional meansas discussed above. The yields of chromanone 7 from the LDA\Mitsunobureaction generally range between about 30% and about 60%, usually about50% based on chromene 4.

Suitable, but non-limiting, examples of azo compounds for the Mitsunobureaction include diethyl azodicarboxylate (DEAD), diisopropylazodicarboxylate (DIAD), dibutyl azodicarboxylate (DBAD),dipiperidinoazodicarboxamide, bis(N⁴-methylpiperazin-1-yl)azodicarboxamide, dimorpholinoazodicarboxamide,and N,N,N',N'-tetramethylazodicarboxamide (TMAD).

Suitable, but non-limiting, examples of phosphorous derivatives for theMitsunobu reaction include triphenylphosphine, tri-n-butylphosphine,triethylphosphine, trimethylphosphine and tris(dimethylamino)phosphine.

Finally, mild borohydride reduction of chromanone 7 in the presence ofCeCl₃ (H₂ O)₇ produced (±)-calanolide A with the desired stereochemicalarrangement. In conducting the reduction reaction, a solution ofchromanone 7 was added dropwise into a solution of reducing agent, e.g.,sodium borohydride and a metal additive, e.g., CeCl₃ (H₂ O)₇ in ethanol.The rate of addition is such that the reaction mixture temperature ismaintained within a range of between about -40° C. and about 60° C.,preferably ranging between about 10° C. and about 30° C. Thereafter, thereaction mixture was stirred at a temperature ranging between about -40°C. and about 60° C.

In general, the amount of metal additive, e.g., CeCl₃ (H₂ O)₇ present inthe reaction mixture ranged between about 0.1 and about 2 mole,preferably ranging between about 0.5 and about 1 mole, per mole ofsodium borohydride. In addition, the amount of sodium reducing agent,e.g., borohydride employed in the reaction generally ranged betweenabout 0.1 and about 12 moles, preferably ranging between about 2 andabout 4 moles, per mole of chromanone 7. Suitable, but non-limiting,examples of reducing agents include NaBH₄ LiAlH₄, (i-Bu)₂ AlH, (n-Bu)₃SnH, 9-BBN, Zn(BH₄)₂, BH₃, DIP-chloride, selectrides and enzymes such asbaker yeast. Suitable, but non-limiting, examples of metal additivesinclude CeCl₃, ZnCl₂, AlCl₃, TiCi₄, SnCl₃, and LnCl₃ and their mixturewith triphenylphosphine oxide. In practicing this invention, sodiumborohydride as reducing agent and CeCl₃ (H₂ O)₇ as metal additive arepreferred.

Thereafter, the reduction mixture was diluted with water and extractedwith a suitable solvent, e.g., ethyl acetate. The extract was dried overa suitable drying agent, e.g., sodium sulfate, and concentrated. Theresulting residue was then purified by conventional means such as silicagel chromatography, using ethyl acetate/hexane solvent mixtures.

Thus, (±)-calanolide A, 1, was successfully prepared with the desiredstereochemical arrangement by treatment of the key intermediate chromene4 with acetaldehyde diethyl acetal or paraldehyde in the presence oftrifluoroacetic acid and pyridine or a two-step reaction including aldolreaction with acetaldehyde and cyclization either under acidic conditionor neutral Mitsunobu condition to produce chromanone 7, followed byLuche reduction via chromanone 7 (see Scheme II).

An alternative route for preparing (±)-calanolide A from chromene 4 wasattempted. A Robinson-Kostanecki reaction on 4 was conducted with sodiumacetate in refluxing acetic anhydride and produced enone 5 in a 65-70%yield (see Scheme II). Enone S, however, failed to afford (±)-calanolideA when being reduced with borohydride reagents and some transition metalreducing agents, presumably because attack at the pyrone and ringopening occurred preferentially. Treatment of compound 5 with Baker'syeast also resulted in coumarin ring cleavage while tri-n-butyltinhydride reduce enone 5 into enol 6 in a modest yield.

In another embodiment of the invention, methods for resolving(±)-calanolide A into its optically active forms, (+)-calanolide A and(-)-calanolide A, are provided. In one method, (±)-Calanolide A isresolved by high performance liquid chromatography (HPLC) with organicsolvent system as a mobile phase. HPLC is performed on a column packedwith chiral packing material. Suitable, but not limiting, examples ofchiral packing material include amylose carbamate, D-phenylglycine,L-phenylglycine, D-leucine, L-leucine, D-naphthylalanine,L-naphthylalanine, or L-naphthylleucine. These materials may be bounded,either ionically or covalently, to silica sphere which particle sizesranging between about 5 μm and about 20 μm.

Suitable, but non-limiting, mobile phase includes hexane, heptane,cyclohexane, ethyl acetate, methanol, ethanol, or isopropanol andmixtures thereof. The mobile phase may be employed in isocratic, stepgradient or continuous gradient systems at flow rates generally rangingbetween about 0.5 mL/min. and about 50 mL/min.

Another method for resolving (±)-calanolide A into its optically activeforms involves enzyme-catalyzed acylation or hydrolysis. In practicingthis invention, enzyme-catalyzed acylation of (±)-calanolide A ispreferred. The enzymatic resolution method employs enzymes such aslipase CC, (Candida Cylindracea), lipase AK (Candida Cylindracea),lipase AY (Candida Cylindracea), lipase PS (Pseudomonas Species), lipaseAP (Aspergillus niger), lipase N (Rhizopus nieveuis), lipase FAP(Rhizopus nieveus), lipase PP (Porcine Pancrease), pig (porcipe) liveresterase (PLE), pig liver acetone powder (PLAP), or subtilisin. Thepreferred enzyme for use in the enzyme-catalyzed acylation reaction islipase PS-13 (Sigma Corporation, St. Louis, Mo., USA). Immobilized formsof the enzyme on cellite, molecular sieves, or ion exchange resin arealso contempated for use in this method. The amount of enzyme used inthe reaction depends on the rate of chemical conversion desired and theactivity of the enzyme.

The enzymatic acylation reaction is carried out in the presence of anacylating agent. Suitable, but not limiting, examples of acylatingagents include vinyl acetate, vinyl propionate, vinyl butyrate, aceticanhydride, propionic anhydride, phthalic anhydride, acetic acid,propionic acid, hexanoic acid or octanoic acid. The enzyme reactionemploys at least one mole of acylating agent per mole of (±)-calanolideA. Acylating agent can be used as a solvent in the acylation reaction oras a co-solvent with another solvent such as hexanes, chloroform,benzene and THF.

One skilled in the art will appreciate that racemic esters of calanolideA can be made by conventional esterification means and selectivelyhydrolyzed by the enzymes so as to produce, in high enantiomeric excess,optically active (+)- or (-)-calanolide A in free or esterified form.The esterified calanolide A may be hydrolyzed chemically orenzymatically into the free form. Suitable, but not limiting examples ofsolvents for use in the enzymatic hydrolysis reaction include water,suitable aqueous buffers such as sodium phosphate buffers or alcoholssuch as methanol or ethanol.

In yet another embodiment of the invention, a method for treating orpreventing viral infections using (±)- and (-)-calanolide A is provided.(±)-Calanolide A and (-)-calanolide A have not been reported before fortheir anti-HIV activity. It has been discovered that (±)-calanolide Ainhibits human immunodeficiency virus type 1 (HIV-1) with EC₅₀ valuebeing approximately half of that for (+)-calanolide A. Although (-)calanolide A is inactive against HIV-1, it does not exhibit synergisticeffect on toxicity of (±)-calanolide A. Therefore, one skilled in theart will appreciate to directly use the synthetic (±)-calanolide A asantiviral agent, without further resolution into the optically pure(+)-calanolide A, to inhibit the growth or replication of viruses in amammal. Examples of mammals include humans, primates, bovines, ovines,porcines, felines, canines, etc. Examples of viruses may include, butnot limited to, HIV-1, HIV-2, herpes simplex virus (type 1 and 2) (HSV-1and 2), varicella zoster virus (VZV), cytomegalovirus (CMV), papillomavirus, HTLV-1, HTLV-2, feline leukemia virus (FLV), avian sarcomaviruses such as rous sarcoma virus (RSV), hepatitis types A-E, equineinfections, influenza virus, arboviruses, measles, mumps and rubellaviruses. More preferably the compounds of the present invention will beused to treat a human infected with a retrovirus. Preferably thecompounds of the present invention will be used to treat a human exposedor infected (i.e., in need of such treatment) with the humanimmunodeficiency virus, either prophylactically or therapeutically.

An advantage of certain compounds of the present invention is that theyretain the ability to inhibit certain HIV RT mutants which are resistantto other non-nucleoside inhibitors such as TIBO and nevirapine orresistant to nucleoside inhibitors. This is advantageous over thecurrent AIDS drug therapy, where biological resistance often develops tonucleoside analogs used in the inhibition of RT.

Hence the compounds of the present invention are particularly useful inthe prevention or treatment of infection by the human immunodeficiencyvirus and also in the treatment of consequent pathological conditionsassociated with AIDS. Treating AIDS is defined as including, but notlimited to, treating a wide range of states of HIV infection: AIDS, ARC,both symptomatic and asymptomatic, and actual or potential exposure toHIV. For example, the compounds of this invention are useful in treatinginfection of HIV after suspected exposure to HIV by e.g., bloodtransfusion, exposure to patient blood during surgery or an accidentialneedle stick.

Antiviral (±)-calanolide A and (-)-calanolide A may be formulated as asolution of lyophilized powders for parenteral administration. Powdersmay be reconstituted by addition of a suitable diluent or otherpharmaceutically acceptable carrier prior to use. The liquid formulationis generally a buffered, isotonic, aqeuous solution. Examples ofsuitable diluents are normal isotonic saline solution, standard 5%dextrose in water or in buffered sodium or ammonium acetate solution.Such formulation is especially suitable for parenteral administration,but may also be used for oral administration. It may be desirable to addexcipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose,acacia, polyethylene glycol, mannitol, sodium chloride or sodiumcitrate.

Alternatively, the compounds of the present invention may beencapsulated, tableted or prepared in an emulsion (oil-in-water orwater-in-oil) syrup for oral administration. Pharmaceutically acceptablesolids or liquid carriers, which are generally known in thepharmaceutical formulary arts, may be added to enhance or stabilize thecomposition, or to facilitate preparation of the composition. Solidcarriers include starch (corn or potato), lactose, calcium sulfatedihydrate, terra alba, croscarmellose sodium, magnesium stearate orstearic acid, talc, pectin, acacia, agar, gelatin, or collodial silicondioxide. Liquid carriers include syrup, peanut oil, olive oil, salineand water. The carrier may also include a sustained release materialsuch as glyceryl monostearate or glyceryl distearate, alone or with awax. The amount of solid carrier varies but, preferably, will be betweenabout 10 mg to about 1 g per dosage unit.

The dosage ranges for administration of antiviral (±)-calanolide A and(-)-calanolide A are those to produce the desired affect wherebysymptoms of infection are ameliorated. For example, as used herein, apharmaceutically effective amount for HIV infection refers to the amountadministered so as to maintain an amount which suppresses or inhibitssecondary infection by syncytia formation or by circulating virusthroughout the period during which HIV infection is evidenced such as bypresence of anti-HIV antibodies, presence of culturable virus andpresence of p24 antigen in patient sera. The presence of anti-HIVantibodies can be determined through use of standard ELISA or Westernblot assays for, e.g., anti-gp120, anti-gp41, anti-tat, anti-p55,anti-p17, antibodies, etc. The dosage will generally vary with age,extent of the infection, the body weight and counterindications, if any,for example, immune tolerance. The dosage will also be determined by theexistence of any adverse side effects that may accompany the compounds.It is always desirable, whenever possible, to keep adverse side effectsto a minimum.

One skilled in the art can easily determine the appropriate dosage,schedule, and method of administration for the exact formulation of thecomposition being used in order to achieve the desired effectiveconcentration in the individual patient. However, the dosage can varyfrom between about 0.001 mg/kg/day to about 50 mg/kg/day, but preferablybetween about 0.01 to about 1.0 mg/kg/day.

The pharmaceutical composition may contain other pharmaceuticals inconjunction with antiviral (±)-calanolide A and (-)-calanolide A, totreat (therapeutically or prophylactically) AIDS. For example, otherpharmaceuticals may include, but are not limited to, other antiviralcompounds (e.g., AZT, ddC, ddI, D4T, 3TC, acyclovir, gancyclovir,fluorinated nucleosides and nonnucleoside analog compounds^(2g) such asTIBO derivatives, nevirapine, pyridinones, BHAP, HEPTs, TSAOS, α-APAα-interferon and recombinant CD4), immunostimulants (variousinterleukins and cytokines), immunomodulators and antibiotics (e.g.,antibacterial, antifungal, anti-pneumocysitis agents). Administration ofthe inhibitory compounds with other anti-retroviral agents that actagainst other HIV proteins such as protease, intergrase and TAT willgenerally inhibit most or all replicative stages of the viral lifecycle.

In addition, the compounds of the present invention are useful as toolsand/or reagents to study inhibition of retroviral reversetranscriptases. For example, the instant compounds selectively inhibitHIV reverse transcriptase. Hence, the instant compounds are useful as astructure-activity relationship (SAR) tool to study, select and/ordesign other molecules to inhibit HIV.

The following examples are illustrative and do not serve to limit thescope of the invention as claimed.

EXPERIMENTAL

All chemical reagents and solvents referred to herein are readilyavailable from a number of commercial sources including Aldrich ChemicalCo. or Fischer Scientific. NMR spectra were run on a Hitachi 60 MHzR-1200 NMR spectrometer or a Varian VX-300 NMR spectrometer. IR spectrawere obtained using a Midac M series FT-IR instrument. Mass spectraldata obtained using a Finnegan MAT 90 mass spectrometer. All meltingpoints are corrected.

EXAMPLE 1

5,7-Dihydroxy-4-propylcoumarin⁵ (2)

Concentrated sulfuric acid (200 mL) was added into a mixture ofphloroglucinol dihydrate (150 g, 0.926 mol) and ethyl butyrylacetate(161 g, 1.02 mol). The resulting mixture was stirred at 90° C. for twohours whereupon it was poured onto ice. The solid product was collectedby filtration, and then dissolved in ethyl acetate. The solution waswashed with brine and dried over Na₂ SO₄. After removal of the solventin vacuo, the residue was triturated with hexane to provide essentiallypure compound 2 (203 g) in quantitative yield, mp 233°-235° C. (Lit.⁵236°-238° C.). ¹ H-NMR⁵ (DMSO-d₆) δ 0.95 (3H,t,J=6.9 Hz, CH₃); 1.63 (2H,apparent hextet, J=7.0 Hz, CH₂); 2.89 (2H,t,J=7.5 Hz,CH₂); 5.85 (1H, s,H₃); 6.22 (1H, d, J=2.0 Hz, H₆); 6.31 (1H, d, J=2.0 Hz, H₈); 10.27 (1H,s, OH); 10.58 (1H, s, OH); MS (EI); 220(100, M+); 205 (37.9, M-CH₃); 192(65.8, M-C₂ H₄); 177 (24.8, M-C₃ H₇); 164 (60.9, M-CHCO₂ +1); 163 (59.6M-CHCO₂); IR (KBr): 3210 (vs and broad, OH); 1649 (vs, sh); 1617 (vs,sh); 1554 (s) cm⁻¹); Anal. calcd. for C,₁₂ H₂₄ O₄ : C, 65.45; H, 5.49;Found: C, 65.61; H, 5.44.

EXAMPLE 2

5,7-Dihydroxy-8-propionyl-4-propylcoumarin (3)

A three-neck flask (500 mL) equipped with an efficient methanicalstirrer, thermometer and addition funnel was charged with5,7-dihydroxy-4-propylcourmarin, 2, (25.0 g, 0.113 mol), aluminumchloride (62.1 g; 0.466 mol), and nitrobenzene (150 mL) and the mixturewas stirred until a solution was obtained, which was cooled to 0° C. inan ice bath. A solution of propionyl chloride (15.2 g; 0.165 mol) incarbon disulfide (50 mL) was added dropwise at such a rate that thereaction temperature was maintained at 8°-10° C. Addition was completedover a period of 1 hour with vigorous stirring. The reaction wasmonitored by TLC using a mobile phase of 50% ethyl acetate/hexane. Afterthree hours, an additional portion of propionyl chloride (2.10 g; 0.0227mol) in carbon disulfide (10 mL) was added. Immediately after the TLCanalysis indicated the total consumption of starting material, thereaction mixture was poured onto ice, and allowed to stand overnight.The nitrobenzene was removed by steam distillation, and the remainingsolution was extracted several times with ethyl acetate. The extractswere combined and dried over. Na₂ SO₄. The crude product obtained byevaporation in vacuo was purified by chromatography on a silica gelcolumn eluting with 50% ether/hexane to provide the desiredpropionylated coumarin 3, mp (corr) 244°-246° C. ¹ H-NMR (DMSO-₆) δ 0.96(3H, t, J=7.3 Hz, CH₃); 1.10 (3H, t, J=7.2 Hz, CH₃); 1.60 (2H, m, CH₂);2.88 (2H, t, J=7.7 Hz, CH₂); 3.04 (2H, q, J=7.2 Hz, CH₂); 5.95 (1H, s,H₃); 6.31 (1H, s, H₆); 11.07 (1H, s, OH); 11.50 (1H, s, OH); MS (EI):277 (6.6, M+1); 276 (9.0, M+); 247 (100, M-C₂ H₅); IR (KBr): 3239 (s andbroad, OH); 1693 (s, C═O), 1625 and 1593 (s) cm⁻¹ ; Anal. calcd. for C₁₅H₁₆ O₅ : C, 65.21; H, 5.84; Found: c, 64.92; H, 5.83. The isomerassignment was made by analogy to precedent.⁷

EXAMPLE 3

2,2-Dimethyl-5-hydroxy-6-propionyl-10-propyl-2H,8H-benzo1,2-b:3,4-b'!dipyran-8-one (4)

A mixture of 3 (2.60 g, 9.42 mmol) and 4,4-dimethoxy-2-methylbutan-2-ol(5.54 g, 37.7 mmol) were dissolved in anhydrous pyridine (6.5 mL). Themixture was refluxed under nitrogen for three days. After removal of thesolvent in vacuo, the residue was dissolved in ethyl acetate. The ethylacetate was washed several times with 1N HCl and brine. It was thendried over Na₂ SO₄. The crude product obtained by evaporation in vacuowas purified by silica gel column chromatography, eluting with 25% ethylacetate/hexane to afford 2.55 g of 4 in 78.6% yield, mp 96°-98° C. ¹H-NMR (CDCl₃) δ 1.05 (3H, t, J=7.3 Hz, CH₃); 1.22 (3H, t, J=7.5 Hz,CH₃); 1.53 (6H, s, 2 CH₃); 1.75 (2H, m, CH₂); 2.92 (2H, t, J=7.1 Hz,CH₂); 3.35 (2H, q, J=7.1 Hz, CH₂); 5.56 (1H, d, J=10.0 Hz, H₃); 5.98(1H, s, H₉); 6.72 (1H, d, J=10.0 Hz, H₄); MS (EI): 343 (5.7, M+1); 342(22.5, M+); 327 (100, M-CH₃); IR (KBr): 1728 (vs, C═O) cm-1; Anal.calcd. for C₂₀ H₂₂ O₅ : C, 70.16; H, 6.48; Found: C, 70.45; H, 6.92.

EXAMPLE 4

10,11-Didehydro-12-oxocalanolide A (5)

A mixture of 4 (1.76 g, 5.11 mmol) and sodium acetate (0.419 g, 5.11mmol) in acetic anhydride (12 mL) were refluxed for 10 hours whereuponthe solvent was removed in vacuo. The residue was purified by silica gelcolumn chromatography, eluting first with 25% ethyl acetate/hexanefollowed by 50% ethyl acetate/hexane to provide 1.16 g (62% yield) ofenone 5 (6,6,10,11-tetramethyl-4-propyl-2H,6H,12H-benzo1,2-b:3,4-b':5,6-b"!-tripyran-2,12-dione) as a white solid, mp209°-209.5° C. ¹ H-NMR (CDCl₃) δ 1.05 (3H, t, J=6.6 Hz, CH₃); 1.56 (6H,s, 2 CH₃); 1.73 (2H, m, CH₂); 1.98 (3H, s, CH₃); 2.38 (3H, s, CH₃); 2.91(2H, t, J=7.5 Hz, CH₂); 5.69 (1H, d, J=10.0 Hz, H₇); 6.11 (1H, s, H₃);6.71 (1H, d, J=10 Hz, H₈); MS (EI): 366 (29.6, M+); 351 (100, M-CH₃);323 (16.5, M-C₃ H₇); IR (KBr): 1734 (vs, C═O), 1657, 1640, 1610, and1562 cm⁻¹ ; Anal. calcd. for C₂₂ H₂₂ O₅ : 72.12; H, 6.05; Found: C,72.14; H, 6.15.

EXAMPLE 5

10,11-Didehydrocalanolide A (6)

A mixture of enone 5 (160 mg, 0.437 mmol) and tri-n-butyltin hydride(0.318 g, 1.09 mmol) in dry dioxane (2.0 mL) was refluxed under nitrogenfor 12 hours. The solvent was then removed in vacuo and the residue waspurified by preparative TLC using 25% ethyl acetate in hexane as themobile phase. The product exhibited an R_(f) of about 0.4. Enol 6(12-hydroxy-6,6,10,11-tetramethyl-4-propyl-2H,6H,12H-benzo1,2-b:3,4-b':970 5,6-b"!-tripyran-2-one) (13.3 mg, 8%) was isolated asan oil from the plate by ethyl acetate elution. This elution may havebeen inefficient, and the actual yield higher, as indicated byanalytical TLC of the crude product. ¹ H-NMR (CDCl₃) δ 0.92 (3H, t,J=6.0 Hz, CH₃); 1.26 (3H, s, CH₃); 1.39 (3H, s, CH₃); 1.63 (2H, m, CH₂);1.96 (3H, s, CH₃); 2.36 (3H, s, CH₃); 2.45 (2H, t, J=6.0 Hz, CH₂); 3.65(1H, s, H₁₂); 5.51 (1H, d, J=10.0 Hz, H₇); 6.67 (1H, d, J=10.0 Hz, H₈);13.25 (1H, br s, OH); MS (EI): 369 (3.8, M+1), 368 (4.4, M+), 367 (8.3,M-1) 366 (28.4, M-2), 351 (100, M-OH); IR(KBr): 1651 (s), 1589 (m)cm⁻¹.

EXAMPLE 6

12-Oxocalanolide A (7)

A solution containing chromene 4 (344 mg, 1.0 mmol), acetaldehydediethylacetal (473 mg, 4.0 mmol), trifluoroacetic acid (1.5 mL, 19.4mmol) and anhydrous pryidine (0.7 mL) was heated at 140° C. under N₂.The reaction was monitored by TLC analysis. After 4 hours, the reactionmixture was cooled to room temperature, diluted with ethyl acetate andwashed several times with 10% aqueous NaHCO₃ and brine. The organiclayer was separated and dried over Na₂ SO₄. The solvent was removed invacuo and the crude product was purified by silica gel columnchromatography eluting with ethyl acetate/hexane (2:3). Chromanone 7(10,11-trans-dihydro-6,6,10,11-tetramethyl-4-propyl-2H,6H,12H-benzo1,2-b:3,4-b':5,6-b"!-tripyran-2,12-dione) (110 mg, 30% yield) wasobtained m.p. 176°˜177° C. (Lit.⁵ 130°˜132° C.). ¹ H-NMR⁵ (CDCl₃) δ 1.02(3H, t, J=7.5 Hz, CH₃); 1.21 (3H, d, J=6.8 Hz, CH₃); 1.51 (3H, d, J=7.0Hz, CH₃); 1.55 (6H, 2s, 2 CH₃); 1.63 (2H, sextet, J=7.0 Hz, CH₂); 2.55(1H, dq, J=6.9 Hz, J=11.0 Hz, H₁₁); 2.88 (2H, t, J=7.6 Hz, CH₂); 4.28(1H, dq, J=6.3 Hz, J=11.0 Hz, H₁₀); 5.60 (1H, d, J=9.9 Hz, H₈); 6.04(1H, s, H₃); 6.65 (1H, d, J=11.8 Hz, H₇); MS (CI): 369 (100, M+1).

EXAMPLE 7

(±)-Calanolide A (1):

To a solution of chromanone 7 (11 mg, 0.03 mmol) in EtOH (0.4 mL) wasadded sodium borohydride (2.26 g, 0.06 mmol) and CeCl₃ (H₂ O)₇ (11.2 mg,0.03 mmol) in EtOH (5 mL) at room temperature. After stirring for 45minutes, the mixture was diluted with H₂ O and extracted with ethylacetate. The organic layer was dried over Na₂ SO₄ and concentrated. Thecrude product was purified by preparative TLC eluting with ethylacetate/hexane (1:1) to afford (±)-calanolide A (1) (10.5 mg, 94%). m.p.52°-54° C., which increased to 102° C. after it was dried thoroughly(Lit.⁵ 56°-58° C.). ¹ H NMR (CDCl₃): δ 1.03 (3H, t, J=7.3 Hz, CH₃) 1.15(3H, d, J=6.8Hz, CH₃), 1.46 (3H, d, j=6.8Hz, CH₃), 1.47 (3H, s, CH₃),1.51 (3H, s, CH₃), 1.66 (2H, m, CH₂), 1.93 (1H, m, H₁₁), 2.89 (2H, m,CH₂), 3.52 (1H, broad-s, OH), 3.93 (1H, m, H₁₀), 4.72 (1H, d, J=7.8 Hz,H₁₂), 5.54 (1H, d, J=10.0 Hz, H₇), 5.94 (1H, s, H₃), 6.62 (1H, d, J=9.9Hz, H₈); MS (CI): 371 (75.4, M+1), 370 (16.1, M⁺), 353 (100, M-OH);Anal. calcd. for C₂₂ H₂₅ O₅ : C, 71.33; H, 7.07: Found: C, 71.63; H,7.21.

EXAMPLE 8

5,7-Dihydroxy-4-propylcoumarin (2):

In this Example, kilogram scale preparation of intermediate 2 isdescribed. Into a stirring suspension of phloroglucinol (3574.8 g, 28.4mol, pre-dried to constant weight) and ethyl butyrylacetate (4600 mL,28.4 mol) was added concentrated sulfuric acid dropwise at such a ratethat the internal temperature did not exceed 40° C. After 100 mL ofsulfuric acid was added, the temperature rose to 70° C. and thesuspension turned into a yellow solid. Analysis of TLC indicated thatthe reaction had proceeded to completion. The reaction mixture wasdiluted with water (10 L) and stirred at ambient temperature overnight.The precipitated product was collected by filtration and then rinsedwith water until the filtrate was neutral. A quantity of 4820 g (77%yield) of 5,7-dihydroxy-4-propylcoumarin 2 was obtained after beingdried, which was identical with an authentic sample by comparsion ofTLC, melting point and spectroscopic data.

EXAMPLE 9

5,7-Dihydroxy-8-propionyl-4-propylcoumarin 3

In this Example, kilogram quantities of intermediate 3 was sythesizedusing propionic anhydride instead of propionyl chloride.5,7-dihydroxy-4-propyl-coumarin, 2, (1710 g, 7.77 mol) and AlCl₃ (1000g, 7.77 mol) were mixed in 1,2-dichloroethane (9 L). The resultingorange suspension was stirred and heated to 70° C. until a solution wasobtained. Then, a mixture of propionic anhydride (1010 g. 7.77 mol) andAlCl₃ (2000 g, 15.54 mol) in 1,2-dichloroethane (3.4 L) was addeddropwise over 3 h. The reaction was allowed to stir at 70° C. for anadditonal hour. After being cooled down to room temperature, thereaction mixture was poured into a rapidly stirring mixture of ice waterand 1N HCl. The precipitated product was taken into ethyl acetate (30 L)and the aqueous solution was extracted with the same solvent (10 L×2).The combined extracts were successively washed with 1N HCl (10 L) ,saturated aq. NaHCO₃ (10 L) , and water (10 L). After being dried overMgSO₄ and concentrated In vacuo, a solid product (1765 g) was obtainedwhich was washed with ethyl acetate (15 L) and recrystallized fromdioxane (9.5 L) to provide 514 g of pure compound 3. From the ethylacetate washings, an additional 100 g of compound was obtained afterrecrystallization from dioxane. Thus, the combined yield for compound 3,which was identical with an authentic sample by comparison of TLC,melting point and spectroscopic data, was 29%.

EXAMPLE 10

2,2-Dimethyl-5-hydroxy-6-propionyl-10-propyl-2H,8H-benzo1,2-b:3,4-b'!dipyran-8-one (4):

In this Example, intermediate 4 was prepared in half kilogram quantitiesfrom 3 via modification of the reaction conditions described in Example3. A mixture of compound 3 (510.6 g, 1.85 mol) and4,4-dimethoxy-2-methylbutan-2-ol (305.6 g, 2.06 mol) were dissolved in amixture of toluene (1.5 L) and dry pyridine (51 mL). This mixture wasstirred and ref luxed; water and MeOH formed during the reaction wereremoved azeotropically via a Dean-Stark trap. The reaction was monitoredby TLC. After 6 days, the reaction had proceeded to completion. Themixture was then cooled to ambient temperature and diluted with ethylacetate (L) and 1N HCl (1 L). The ethyl acetate solution was separatedand washed with 1N HCl (500 mL) and brine (1 L). After being dried overNa₂ SO₄ and evaporated in vacuo, a quantity of 590 g (93% yield) ofcompound 4 was obtained which was greater than 95% pure without furtherpurification and was compared with an authentic sample by TLC andspectroscopic data. No trace of 6-acylated or 6,8-bisacylated productwas observed, although a small amount of 7-monoester did form.

EXAMPLE 11

12-Oxocalanolide A (7):

In this Example, chromanone 7 was prepared from two alternative pathwaysinvolving either a one-step reaction (procedure A) or a two-stepreaction process (procedures B and C).

Procedure A. Paraldehyde One-Step Reaction: To a stirring solution ofchromene 4 (350 mg, 1.0 mmol) and PPTS (250 mg, 1.0 mmol) in1,2-dichloroethane (2 mL) at ambient temperature under N₂ was added 3 mLparaldehyde (22.5 mmol). The resulting mixture was ref luxed for 7 h.Then, CF₃ CO₂ H (1 mL), an additional equivalent of PPTS and 1 mL ofparaldehyde were added; the mixture was refluxed overnight. The reactionmixture was neutralized with saturated aqueous NaHCO₃ and extracted withethyl acetate (50 mL×3). The crude product obtained by evaporation underreduced pressure was washed with hexane. The residue was purified bycolumn chromatography eluting with ethyl acetate/hexane (1:2) to afford100 mg (27% yield) of chromanone 7 and 30 mg (8% yield) of 7a.Chromanone 7(10,11-trans-dihydro-6,6,10,11-tetramethyl-4-propyl-2H,6H,12H-benzo1,2-b:3,4-b':5,6-b"!tripyran-2,12-dione) obtained by this method wasidentical with an authentic sample by comparison of TLC, HPLC andspectroscopic data.

Procedure B LDA/Sulfuric Acid Two-Step Reaction: To a stirring solutionof chromene 4 (5.0 g, 14.6 mmol) in THF (75 mL) at -30° C. under N₂ wasadded 18.3 mL (36.5 mmol) of 2M LDA in THF. After 15 min at the sametemperature, acetaldehyde (5.0 mL, 89.5 mmol) was added via syringe. Thereaction was monitored by TLC analysis. After 1 h, the reaction mixturewas quenched at -10° C. with saturated aqueous NH₄ Cl (75 mL) andextracted with ethyl acetate (125 mL×3). The combined extracts werewashed with brine (125 mL) and dried over Na₂ SO₄. Removal of solventsin vacuo afforded a reddish oil of 7b (8.5 g).

The crude 7b was dissolved in acetic acid (100 mL) and then 50% H₂ SO₄(100 mL) was added with stirring. The resulting mixture was heated at75° C. for 2.5 h and then at 50° C. for 4 h. TLC analysis indicated thatthe starting material had been consumed. The reaction mixture wasdetermined to contain both chromanone 7 and 10,11-cis-dimethylderivative 7a in a 1:1 ratio. After cooling to ambient temperature, thereaction mixture was poured into a mixture of ice water (500 mL) andethyl acetate (500 mL). The layers were separated and the aqueous layerwas extracted with ethyl acetate (200 mL×3). The ethyl acetate solutionswere combined and washed with saturated aqueous NaHCO₃ and brine. Afterbeing concentrated in vacuo, the product was purified by chromatographyon a silica gel column eluting with ethyl acetate/hexane (2:3) toprovide 850 mg (16% yield) of chromanone 7, which was further purifiedby recrystallization from ethyl acetate/hecane and was identical with anauthentic sample by comparison of TLC, HPLC and spectroscopic data.

Procedure C. LDA/Mitsunobu Two-Step Reaction: Into a stirring solutionof THF (10 mL) containing triphenylphosphine (1.27 g, 4.80 mmol) and thecrude 7b, obtained from chromene 4 (1.0 g, 2.34 mmol), 2.5 equivalentsof LDA and 6.0 equivalents of acetaldehyde by the procedure describedabove, was added dropwise diethyl azodicarboxylate (DEAD, 0.77 mL, 4,89mmol). The resulting reddish solution was stirred at ambient temperatureunder N₂ for 1 h, after which the reaction mixture was quenched withsaturated aqueous NH₄ Cl and extracted with ethyl acetate (50 mL×3). Theextracts were washed with brine and dried over Na₂ SO₄. After removal ofsolvents, the crude product was purified by column chromatography onsilica gel eluting with ethyl acetate/hexane (2:3) to provide 412 mg(48% yield, based on chromene 4) of chromanone 7, the predominantproduct of the reaction, which was identical with an authentic sample bycomparison of TLC, HPLC and spectroscopic data.

EXAMPLE 12

(±)-Calanolide A (1):

In this Example, (±)-calanolide A was prepared in multi-gram scale usingthe procedure described in Example 7. To a stirring solution ofchromanone 7 (51.5 g, 0.14 mol) in EtOH (1.5 L) was added CeCl₃ (H₂ O)₇(102 g, 274 mmol). The mixture was stirred for 1.5 h at room temperatureunder N₂ and then cooled to -30° C. with an ethylene glycol/H₂ O (1:2w/w) dry ice bath. After the temperature was equilibrated to -30° C.,NaBH₄ (21.3 g, 563 mmol) was added and stirred at the same temperaturefor 8.5 h, at which time the reaction was quenched with H₂ O (2 L) andextracted with ethyl acetate (2 L×3). The extracts were combined, washedwith brine (2 L) and dried over Na₂ SO₄. The crude product obtained byremoval of solvent under reduced pressure was passed through a shortsilica gel column to provide 53 g of mixture which contained 68% of(±)-calanolide A, 14% of calanolide B and 13% of chromanone 7 as shownby HPLC. This material was subjected to further purification bypreparative HPLC to afford pure (±)-calanolide A (1).

EXAMPLE 13

Chromatographic Resolution of Synthetic (±)-Calanolide A

The synthetic (±)-1 was resolved into enantiomers, (+)-calanolide A and(-) calanolide A, by preparative HPLC. Thus, using a normal phase silicagel HPLC column (250 mm×4.6 mm I.D. Zorbasil, 5 μm particle size,MAC-MOD Analytical, Inc., P., USA), the synthetic (±)-1 appeared as onepeak with a retention time of 10.15 minutes when hexane/ethyl acetate(70:30) was used as the mobile phase at a flow rate of 1.5 mL/min and awavelength of 290 nm was used as the uv detector setting. However, on achiral HPLC column packed with amylose carbamate (250 mm×4.6 mm I.D.Chiralpak AD, 10 μm particle size, Chiral Technologies, Inc., P., USA),two peaks with retention times of 6.39 and 7.15 minutes in a ratio of1:1 were observed at a flow rate of 1.5 mL/min. The mobile phase washexane/ethanol (95:5) and the uv detector was set at a wavelength of 254nm. These two components were separated using a semi-preparative chiralHPLC column, providing the pure enantiomers of calanolide A. Thechemical structures of the separated enantiomers, which were assignedbased on their optical rotations and compared with the reported naturalproduct, were characterized by spectroscopic data. HPLC chromatograms(±)-calanolide A and its optical forms are shown in FIG. 6.

(±)-Calanolide A (1): mp 47°-50° C. (Lit.¹⁴ 45°-48° C. ); α!²⁵ _(D)=+68.8° (CHCl₃, c 0.7) (Lit.¹⁴ α!²⁵ _(D) =+66.6° (CHCl₃, c 0.5); ¹ H NMR(CDCl₃) δ 1.03 (3H, t, J=7.3 Hz, CH₃), 1.15 (3H, d, J=6.8 Hz, CH₃), 1.46(3H, d, J=6.4 Hz, CH₃), 1.47 (3H, s, CH₃), 1.51 (3H, s, CH₃), 1.66 (2H,m, CH₂), 1.93 (1H, m, H₁₁), 2.89 (2H, m, CH₂), 3.52 (1H, d, J=2.9 Hz,OH), 3.93 (1H, m, H₁₀), 4.72 (1H, dd, J=7.8 Hz, J=2.7 Hz, H₁₂), 5.54(1H, d, J=9.9 Hz, H₇), 5.94 (1H, s, H₃), 6.62 (1H, d, J=9.9 Hz, Hg); ¹³C NMR (CDCl₃) δ 13.99 (CH₃), 15.10 (CH₃), 18.93 (CH₃), 23.26 (CH₂),27.38 (CH₃), 28.02 (CH₃), 38.66 (CH₂), 40.42 (CH), 67.19 (CH--OH), 77.15(CH--O), 77.67 (C--O), 104.04 (C_(4a)), 106.36 (C_(8a) and C_(12a)),110.14 (C₃), 116.51 (C--O), 126.97 (C₇), 151.14 (C_(4b)), 153.10(C_(8b)), 154.50 (C_(12b)), 158.88 (C₄), 160.42 (C═O); CIMS: 371 (100,M+1), 370 (23.6,M⁺), 353 (66.2, M-OH); 1R: 3611 (w) and 3426 (m, broad,OH), 1734 (vs. C═O), 1643 (m), 1606 (m) and 1587 (vs) cm⁻¹ ; UV λ_(max)(MeOH): 204 (32,100), 228 (23,200), 283 (22,200), 325 (12,700) nm; Anal.calcd. for C₂₂ H₂₆ O ₅ 1/4H₂ O: C, 70.47; H, 7.12; Found: C, 70.64; H,7.12.

(-)-Calanolide A (1): mp 47°-50° C.; α!^(25D) =+75.6° (CHCl₃, c 0.7)Lit.¹⁴ α!^(25D) =-66° (CHCl₃, c 0.5); ¹ H NMR (CDCl₃) δ 1.03 (3H, t,J=7.4 Hz, CH₃), 1.15 (3H, d, J=6.8 Hz, CH₃), 1.46 (3H, d, J=6.3 Hz,CH₃), 1.47 (3H, s, CH₃), 1.51 (3H, s, CH₃), 1.66 (2H, m, CH₂), 1.93 (1H,m, H₁₁), 2.89 (2H, m, CH₂), 3.50 (1H, d, J=2.9 Hz, OH), 3.92 (1H, m,H₁₀), 4.72 (1H, dd, J=7.8 Hz, J=2.7 Hz, H₁₂), 5.54 (1H, d, J=10.0 Hz,H₇), 5.94 (1H, S, H₃), 6.62 (1H, d, J=10.0 Hz, H₈); ¹³ C NMR (CDCl₃) δ13.99 (CH₃), 15.10 (CH₃), 18.93 (CH₃), 23.36 (CH₂), 27.38 (CH₃), 28.02(CH₃), 38.66 (CH₂), 40.42 (CH), 67.19 (CH--OH), 77.15 (CH--O), 77.67(C--O), 104.04 (C_(4a)), 106.36 (C_(8a) and C_(12a)), 110.14 (C₃),116.51 (C₈), 126.97 (C₇), 151.14 (C_(4b)), 153. 11 (C_(8b)), 154.50(C_(12b)), 158.90 (C₄), 160.44 (C═O); CIMS: 371 (95.2, M+1), 370(41.8,M⁺), 353 (100, M-OH); IR: 3443 (m, broad, OH), 1732 (vs, C═O),1643 (m), 1606 (m) and 1584 (vs) cm⁻¹ ; UV λ_(max) (MeOH): 200 (20,500),230 (19,400), 283 (22,500), 326 (12,500) nm; Anal. calcd. for (C₂₂ H₂₆O₅ 1/4H₂ O: C, 70.47; H, 7.12; Found: C, 70.27; H, 7.21.

EXAMPLE 14

Enzymatic Resolution of (±)-Calanolide A

To a magnetically stirred suspension of (±)-Calanolide A, prepared bythe method of the present invention, and vinyl butyrate (0.1 mL) inhexane (0.5 mL) at ambient temperature was added 1 mg of lipase PS-13(Pseudomonas Species) (Sigma Corporations, St. Louis, Mo., USA). Thereaction mixture was stirred and monitored by conventional means such asTLC analysis. At 10 days, an additional 1 mg of lipase PS-13 was added.After stirring for a total of 20 days, the reaction was stopped becausethere was no obvious increase in ester formation. The enzyme wasfiltered out and the filtrate was concentrated to dryness. The residuewas analyzed by HPLC (see Example 13), which showed that 21% of(-)-calanolide A had been converted into its butyrate ester form. Theenriched (+)-calanolide A and the butyrate ester of (-)-calanolide A canbe easily separated by conventional means such as column chromatography.The enriched (+)-calanolide A may be repeatedly treated with vinylbutyrate and lipase PS-13 as described above so as to obtain high e.e.of (+)-calanolide A.

EXAMPLE 15

In Vitro evaluation of (±)- and (-)-calanolide A

This example illustrates the anti-HIV viral activity of the synthetic(±)-calanolide A and its pure enantiomers, (+)-calanolide A and(-)-calanolide A, were evaluated using the published MTT-tetrazoliummethod.²⁰ Retroviral agents, AZT and DDC, were used as controls forcomparison purposes.

The cells used for screening were the MT-2 and the humanT4-lymphoblastoid cell line, CEM-SS, and were grown in RPMI 1640 mediumsupplemented with 10% fetal (v/v) heat-inactivated fetal calf serum andalso containing 100 units/ml penicillin, 100 μg/ml streptomycin, 25 mMHEPES and 20 μg/ml gentamicin. The medium used for dilution of drugs andmaintenance of cultures during the assay was the same as above. TheHTLV-IIIB and HTLV-RF were propagated in CEM-SS. The appropriate amountsof the pure compounds for anti-HIV evaluations were dissolved in DMSO,then diluted in medium to the desired initial concentration. Theconcentrations (ug drug/mL medium) employed were 0.0032 ug/mL; 0.001ug/ml; 0.0032 ug/mL; 0.01 ug/mL; 0.032 ug/mL; 0.1 ug/mL; 0.32 ug/mL; 1ug/mL; 3.2 ug/mL; 10 ug/mL; 32 ug/mL; and 100 u g/mL. Each dilution wasadded to plates in the amount of 100 μl/well. Drugs were tested intriplicate wells per dilution with infected cells while in duplicatewells per dilution with uninfected cells for evaluation of cytotoxicity.On day 6 (CEM-SS cells) and day 7 (MT-2 cells) post-infection, theviable cells were measured with a tetrazolium salt, MTT (5 mg/ml), addedto the test plates. A solution of 20% SDS in 0.001N HCl is used todissolve the MTT formazan produced. The optical density value was afunction of the amount of formazan produced which was proportional tothe number of viable cells. The percent inhibition of CPE per drugconcentration was measured as a test over control and expressed inpercent (T/C %). The data is summarized in FIGS. 1(a-e), 2(a-e), 3(a-e),4(a-d), and 5(a-d).

FIGS. 1(a) to 1(e) illustrate in vitro MTT assay results using anisolate, G9106 HIV viral strain²¹, which is AZT-resistant. The datashows that (-)-calanolide A was relatively non-toxic at concentrationsof 1 ug/mL but exhibited very little antiviral effect. Moreover,(±)-calanolide A was effective as (+)-calanolide A in reducing viralCPE. As expected, AZT had little to no effect in reducing viral CPE andenhancing cell viability.

FIGS. 2(a) to 2(e) illustrate in vitro MTT assay results using H112-2HIV viral strain which was not pre-treated with AZT. As expected, theviral strain was sensitive to AZT. The data also showed that(-)-calanolide A was relatively non-toxic at concentrations of 1 ug/mLbut exhibited very little antiviral effect. (±)-calanolide A was nearlyas effective as (+)-calanolide A in reducing viral CPE.

FIGS. 3(a) to 3(e) illustrate in vitro MTT assay results using A-17 HIVviral strain²² which is resistant to to non-nucleoside inhibitors suchas TIBO and pyridinone but is sensitive to AZT. The results hereparallel those shown in FIGS. 2(a)-2(e).

FIGS. 4(a)-(e) and 5(a)-(e) illustrate in vitro MTT assay results usinglab cultivated HIV viral strains IIIB and RF, respectively. The resultshere also parallel those shown in FIGS. 2(a)-2(e).

REFERENCES:

1a. Brookmeyer, R., Reconstruction and Future Trends of the AIDSEpidemic in the United States, Science, 1991, 253, 37-42.

b. Brain, M. M.; Heyward, W. L.; Curran, J. W., The Global Epidemiologyof HIV Infection and AIDS, Annu. Rev. Microbiol., 1990, 44, 555-577.

2a. Weislow, O. S.; Kiser, R.; Fine, D. L.: Bader, J. Shoemaker, R. H.;Boyd, M. R., New Soluble-formazan Assay for HIV-1 Cytopathic Effects:Application to High-Flux Screening of Synthetic and Natural Products ofAIDS-Antiviral Activity. J. Natl. Cancer Inst., 1989, 81, 577-586.

b. Mitsuya, H.; Yarchoan, R.; Broder, S., Molecular Targets for AIDSTherapy. Science, 1990, 249, 1533-1544.

c. Petteway, S. R., Jr.; Lambert, D. M.; Metcalf, B. W., The ChronicallyInfected Cells: A Target for the Treatment of HIV Infection and AIDS.Trends Pharmacol. Sci., 1991, 12, 28-34.

d. Richman, D. D., Antiviral Therapy of HIV Infection, Annu. Rev. Med.,1991, 42, 69-90.

e. Haden, J. W., Immunotherapy of Human Immunodeficiency VirusInfection. Trends Pharmacol Sci., 1991, 12, 107-111.

f. Huff, J. R., HIV Protease: A Novel Chemotherapeutic Target for AIDS.J. Med. Chem., 1991, 34, 2305-2314.

g. De Clercq, E., HIV Inhibitors Targeted at the Reverse Transcriptase.AIDS Research and Human Retroviruses, 1992, 8, 119-134.

3. Kashman, Y.; Gustafson, K. R.; Fuller, R. W.; Cardellina, J. H., II;McMahon; J. B.; Currens, M. J.; Buckheit, R. W., Jr.; Hughes, S. H.;Cragg, G. M.; Boyd, M. R., The Calanolides, a Novel HIV-Inhibitory Classof Coumarin Derivatives from the Tropical Rainforest Tree, Calophyllumlanigerum. J. Med. Chem. 1992, 35, 2735-2743.

4. Boyd, M. R., National Cancer Institute, Personal Communication.

5. Chenera, B.; West, M. L.; Finkelstein, J. A.; Dreyer, G. B., TotalSynthesis of (±)-Calanolide A, a Non-Nucleoside Inhibitor of HIV-1Reverse Transcriptase. J. Org. Chem. 1993, 58, 5605-5606.

6. Sethna, S.; Phadke, R., The Pechmann Reaction. Organic Reactions,1953, 7, 1-58 and references cited therein.

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8. Barton, D. H. R.; Donnelly, D. M. X.; Finet, J. P.; Guiry, P. J.,Total synthesis of Isorobustin. Tetrahedron Lett. 1990, 31, 7449-7452.

9. Kovacs, T. S.; Zarandy, M. S.; Erdohelyi, A., Cyclization of the EnolEsters of o-Acyloxyphenyl Alkyl Ketones, IV. A Kenetic Study of theSteps of the Kostanecki-Robinson Reaction. Helv. Chim. Acta, 1969, 52,2636-2641.

10. Fung, N. Y. M.; de Mayo, P.; Schauble, J. H.; Weedon, A. C,Reduction by Tributyltin Hybride of Carbonyl Compounds Absorbed onSilica Gel: Selective Reduction of Aldehydes, J. Org. Chem. 1978, 43,3977-3979.

11. Hughes, D. L., The Mitsunobu Reaction. Organic Reaction, 1992, 42,335-656 and references cited therein.

12. Gemal, A. L.; Luche, J. L., Lanthanoids in organic Synthesis. 6. TheReduction of α-Enones by Sodium Borohydride in the Presence ofLanthanoid Chlorides: Synthetic and Mechanistic Aspects. J. Am. Chem.Soc., 1981, 103, 5454-5459.

13. Very recently, a similar work has been published in the literature;Cardellina, J. H., II; Bokesch, H. R.; McKee, T. C.; Boyd, M. R.,Resolution and Comparative Anti-HIV Evaluation of the Enantiomers ofCalanolides A and B. Bioorg. Med. Chem. Lett. 1995, 5, 1011-1014.

14. Deshpande, P. P., Tagliaferri, F.; Victory, S. F.; Yan, S.; Baker,D. C., Synthesis of Optically Active Calanolides A and B. J. Org. Chem.1995, 60, 2964-2965.

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16. For reviews, see:

(a) Mukaiyama, T., The Directed Aldol Reaction. Org. React. 1982, 28,203-331.

(b) Reetz, M. T., Chelation or Non-Chelation Control in AdditionReactions of Chiral α- and β-Alkoxy Carbonyl Compounds, Angew. Chem.Int. Ed. Eng. 1984, 23, 556-569.

(c) Shibata, I.; Baba, A., Organotin Enolates in organic Synthesis. Org.Prep. Proc. Int. 1994, 26, 85-100.

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What is claimed is:
 1. A method for preparation of chromanone 7 ##STR4##comprising cyclization of aldol product 7b ##STR5## in the presence ofan acid or in the presence of an azo compound and a phosphorusderivative.
 2. The method of claim 1, wherein the acid comprisessulfuric acid, hydrochloric acid, trifluoroacetic acid, methanesulfonicacid, trifluoromethanesulfonic acid, p-tosylic acid, acetic acid ormixtures thereof.
 3. The method of claim 1, wherein the azo compoundcomprises diethyl azodicarboxylate, diisopropyl azodicarboxylate,dibutyl azodicarboxylate, dipiperidinoazodicarboxamide, bis(N⁴-methylpiperazin-1-yl)azodicarboxamide, dimorpholinoazodicarboxamide, orN,N,N',N'-tetramethylazodicarboxamide.
 4. The method of claim 1, whereinthe phosphorous derivative comprises triphenylphosphine,tri-n-butylphosphine, triethylphosphine, trimethylphosphine andtris(dimethylamino)phosphine.
 5. A method of preparation of chromanone 7##STR6## comprising treating cis-chromanone 7a ##STR7## with a base soas to form chromanone
 7. 6. The method of claim 5, wherein the basecomprises a metal hydroxide, a metal alkoxide, a metal hydride, a metalamide, an amine, or LiHMDS.